International Journal of Agricultural Sciences

DOI:10.15740/HAS/IJAS/13.1/71-76
Volume 13 | Issue 1 | January, 2017 | 71-76  e ISSN–0976–5670 Visit us : www.researchjournal.co.in

RESEARCH PAPER

Optimization of explants density for tissue culture

propagation of banana cv. ‘GRANDE NAINE’

G. PRABHULING* AND B.N. SATHYANARAYANA1

Center for Horticulture Biotechnology, Directorate of Research, University of Horticultural Sciences,
BAGALKOT (KARNATAKA) INDIA (Email: gprabhuling@gmail.com)

Abstract : Cost of production is always stressed as the main obstacle for tissue culture. Nutrient media is one of the most costly
input which accounts for 30-35 per cent of total cost of tissue culture propagation. Production cost, therefore, can be reduced by
efficient utilization of culture media. To find out optimum quantity of media required for shoot proliferation, 1- 5 multiple bud
explants were incubated in each culture bottle. Explants density of 4/culture bottles was found best as it recorded higher total
shoot production/l (291.94), shoot length (3.22 cm), number of leaves/shoot (2.81) and lower cost per shoot (Rs. 1.175). In vitro
rooting was carried out with densities of 6, 8, 10 and 12 microshoots/culture bottle. The maximum response with regard to rooting
had not yet been reached as there were no significant differences among the treatments. Incubation of 4 multiple bud explants and
12 microshoots per culture bottles is optimum for scaling-up of tissue culture production of banana cv. ‘ GRANDE NAINE’.
Key Words : Tissue culture, Explants density, Grande Naine, MS medium, Cost/shoot

View Point Article : Prabhuling, G. and Sathyanarayana, B.N. (2017). Optimization of explants density for tissue culture propagation of
banana cv. ‘GRANDE NAINE’. Internat. J. agric. Sci., 13 (1) : 71-76, DOI:10.15740/HAS/IJAS/13.1/71-76.
Article History : Received : 17.10.2016; Revised : 14.11.2016; Accepted : 14.12.2016

INTRODUCTION is one of the most costly input which accounts for 30-35
per cent of total cost of tissue culture propagation. The
Edible bananas (Musa spp.) are among the most
production cost can be reduced by resorting to optimum
important food crops in the world, with a production
explants density during micropropagation. Explant density
approximating 102 million tons per year Anonymous
is an important physical parameter that influences
(2002). However, expansion of banana production is
microplant growth. The need for an optimum explants
limited by lack of availability of healthy and good quality
density for faster micro plant growth during
planting material. The transmission of harmful insects,
micropropagation has been reported in a number of crop
nematodes, viruses and disease by field grown suckers
species (Chun et al., 1986; Hamad and Taha, 2009;
prompted interest in the use of in vitro techniques.
Monette, 1983 and Start and Cumming, 1976).
However, the production cost of micropropagated
Although, easy to test and simple to apply, the effect
plantlets is very high, making small-scale farmers
of explants density on the in vitro multiplication of banana
reluctant to use these superior plantlets. Nutrient media
was totally ignored in the previous studies. In most of
* Author for correspondence:
1
Division of Horticulture, University of Agricultural Sciences, G.K.V.K., BENGALURU (KARNATAKA) INDIA
 G. PRABHULING AND B.N. SATHYANARAYANA
(i)
(ii)
Fig. A : Initiation of aseptic culture in banana cv. GRANDE NAINE by shoot-tip culture (i) Sword suckers; (ii - iii) Shoot-tip culture;
(iv) Aseptic culture
the studies, the attention was focused on optimization of
hormones, carbon sources and gelling agents. Treatment
with highest shoot formation/explant was considered
optimal and suggested for large scale production of
propagules. However, higher shoot formation/single
explant is not the best indicator for commercial large
scale production. The total shoot production and cost/
single shoot are the most important and essential
parameters. The present study was, therefore, conducted
with the objective to compare the effect of explant density
on average shoot formation, shoot length, root formation,
expected total shoots/l medium, the cost of single shoot
and rooted plantlets.
MATERIAL AND METHODS
Healthy and vigorously growing sword suckers of
banana cv. GRANDE NAINE (3-4 month), free from
disease were selected as a source of explant (Fig. A i).
The procedure described by Besagarahally (1996) was
followed for initiation of aseptic culture with certain
modifications.
Preparation of explants :
The plant material obtained from the field was
thoroughly washed in running tap water followed by
washing with a detergent solution to remove adhering
soil particles. Later, rhizomes were kept immersed in 1
per cent (w/v) Bavistin for half an hour, to further clean
the suckers. The outer leaves, leaf base and corm tissue
Internat. J. agric. Sci. | Jan., 2017 | Vol. 13 | Issue 1 | 71-76
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(iii) (iv)
were trimmed using a sterilized stainless steel knife until
the length of explant was 4-6 cm and the diameter, 3-4
cm. These trimmed suckers enclosing the shoot tip were
washed with double distilled water. After trimming one
more outer layer, they were soaked in a solution of 0.5
per cent (w/v) Bavistin + 0.05% (w/v) Streptocycline
for eight hours. After thoroughly washing with double
distilled water, they were trimmed again, so that trimmed
suckers were of 2-3 cm in length and 2-2.5 cm in
diameter. These shoot tips were soaked in 0.05 per cent
(w/v) cetrimide for 30 minutes. After removing one more
layer, the shoot tips were surface sterilized with 0.1 per
cent HgCl2 for 10 minutes. Further operations such as
washing several times with sterile distilled water to
remove all traces of chlorine, trimming of explant and
inoculation in liquid culture media were carried out under
a laminar air flow chamber.
Initiation of aseptic culture :
Shoot tip explants were incubated in MS liquid culture
media containing 2 mg/lit BAP and 75 mg/lit adenine
sulphate for two weeks maintaining standard culture
conditions of 25 ± 20 C, 70 per cent RH and photoperiodic
cycle of 16 hours light and 8 hours dark period (Fig. Aii-
iii). After two weeks of incubation, all the explants (Fig.
A iii) were evaluated for their ability to establish in liquid
media. Greening and swelling of the explants were utilized
as important criteria for assessing the success in
establishment. Shoot tips that had turned dark brown/
Hind Agricultural Research and Training Institute
 OPTIMIZATION OF EXPLANTS DENSITY FOR TISSUE CULTURE PROPAGATION OF BANANA CV. ‘GRANDE NAINE’

black and which did not swell were considered as non-
established. Healthy and contaminant free explants
were excised by removing discoloured tissue and
transferred to the semi-solid media supplemented with
2 mg/lit BAP and 75 mg/lit adenine sulphate and
incubated for four weeks maintaining standard culture
conditions. The explants were observed for their
bulging in the tips and morphogenetic activity. The
successfully established explants (Fig.A iv) were
excised by trimming the discoloured tissues, then 2-4
vertical cuts were made at the tip of each explant and
the used for the experiments.
Baby jar glass bottle (11 cm x 5.5 cm) each with
40 ml MS semisolid medium and multiple bud explants
each having 2 to 3 growing buds were used for studies.
For shoot proliferation explants density of 1, 2, 3, 4 and 5
multiple bud explants were inoculated per culture bottles.
Explants were incubated for eight weeks (transferred to
fresh media after four weeks) maintaining standard
culture conditions. Individual microshoots were obtained
by cutting the multiple shoot clumps using sterilized
scalpel and cultured on rooting medium containing 2
mg/lit IBA +1 mg/lit NAA + activated charcoal 2.5 g/
l for four weeks. At the end of experiments,
morphological characteristics (number of shoots/
explant, shoot length, number of leaves, roots/shoots,
fresh weight etc.,) were measured. An analysis of
variance (ANOVA) was conducted on data concerning
shoot and root morphological parameters using the
statistical programme wax vms fortran.
RESULTS AND DISCUSSION
It is evident (Table 1) that with increase in the
explants density from 2 to 5/ culture bottles, the capacity
of the shoot formation, shoot length and number of
leaves/shoots were reduced (11.33 to 6.98; 3.77 to 2.37
cm and 3.53 to 2.29). Increasing the density of explants/
culture bottles from 3 to 5 resulted in significantly higher
total shoots/litre of medium (262.44, 291.94 and 312.94)
than using 1 and 2 explants/culture bottles (195.56 and
202.25).The cost/shoot was reduced from 1.754 and
1.696 rupees at density of 1 and 2 explants to 1.307 to
1.096 rupees at density of 3 to 5 explants/culture bottles.
The cost per single shoot as well as the total shoots
production and rate of shoot formation per explant of
‘Grand Naine’ banana was significantly affected by the
explants density (Table 1 and Fig. 1). The results indicated
the cost/shoot could be reduced to as low as 1.307 to
1.096 rupees by incubating 4 to 5 explants/culture bottle.
Assessment based on shoot formation/explant is not
enough for selection of best treatment. According to shoot
formation/single explant, density of 1 explant/culture bottle
would be recommended over higher density. However,
compared to density of 1 explant/culture bottle, density
of 4 and 5 culture bottles increased the total shoot
production and reduced the cost per shoots (Table1 and
Fig. 1).
Cost of production always stressed as the main
obstacle of micropropagation. Nevertheless, the cost of
items such as medium volume and medium use efficiency
(total shoot/ litre), autoclaving and laminar operation time,
labour working hours and shelving space, was not taken
into consideration during assessment of different in vitro
multiplication treatments. All these cost items are related
and could be managed through selection of optimum
density of explants/culture. The cost of 1 lit of medium
used as a basis to compare the cost effectiveness of
different cytokinins on banana shoots formation
(Arinaitwe et al., 2000). Using the same approach Goel
et al. (2007) reported that cost/shoot of Rauwolfia
serpentina on semi-solid medium was Rs. 0.126 and
could be reduced to Rs. 0.004 using glass beads with
Daurala sugar. Similar findings were also reported by

Table 1: Effect of explants density on shoot formation in banana cv. GRANDE NAINE
Explants/ Shoots/ Shoot length Shoot diameter Number of Fresh Wt. of Total shoots/ Cost/shoot
bottles (No.) explants (cm) (mm) leaves/shoot shoots (mg) litre medium (Rs.)

1 21.90 3.59 4.00 3.08 641.25 195.56 1.754

2 11.33 3.77 3.75 3.53 612.50 202.25 1.696

3 9.80 3.41 3.50 3.00 551.50 262.44 1.307

4 8.18 3.22 3.00 2.81 357.00 291.94 1.175

5 6.98 2.37 1.77 2.29 291.25 312.94 1.096

C.D.(P=0.01) 7.14 0.65 1.03 0.45 66.26 116.62 ----

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73 Hind Agricultural Research and Training Institute
 G. PRABHULING AND B.N. SATHYANARAYANA

A B

C D

E F

G H

I J
Fig. 1 : Multiple bud clump and microshoots obtained with (A) – (B) 1 explant/culture bottles; (C) – (D) 2 explants/culture
bottles; (E) – (F) 3 explants/culture bottles; (G) –(H) 4 explants/culture bottles; (I) –(J) 5 explants/culture bottles

Internat. J. agric. Sci. | Jan., 2017 | Vol. 13 | Issue 1 | 71-76

74 Hind Agricultural Research and Training Institute
 OPTIMIZATION OF EXPLANTS DENSITY FOR TISSUE CULTURE PROPAGATION OF BANANA CV. ‘GRANDE NAINE’

Fig. 2 : In vitro rooted plantlets obtained with (A) 6 microshoots/culture bottles; (B) 8 microshoots/culture bottles; (C) 10
microshoots/culture bottles; (D) Twelve microshoots/culture bottles

Table 2: Effect of microshoot density on root formation in banana cv. GRANDE NAINE
Number of Number of Root Number of Plant Fresh Cost/
microshoots/ primary length secondary diameter weight plantlets
bottles (No.) roots/shoots (cm) roots/shoot (mm) of plants (mg) (Rs.)
6 5.29 4.77 10.08 3.35 2132.50 2.285
8 5.69 4.69 10.37 3.10 1827.50 1.714
10 4.87 4.66 13.40 3.78 2282.50 1.371
12 4.98 3.80 7.23 4.47 1547.50 1.143
C.D.(P=0.01) 1.97 1.45 5.38 1.40 770.56 -----

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75 Hind Agricultural Research and Training Institute
 G. PRABHULING AND B.N. SATHYANARAYANA
Hamad and Taha (2009), who observed that the cost/
shoots of pineapple could be reduced from 0.13 ringgit
to 0.10 ringgit (Malaysian currency).
Maximum number of secondary roots/shoots and
fresh weight/plantlets were observed when there were
10 microshoots/culture bottles (Table 2 and Fig. 2).
However, the maximum response with regard to root
characters had not yet reached. Therefore, more than
12 microshoots/ culture bottles might produce a greater
response.
The shoot and root growth during the
micropropagation appears to depend on explant density.
The enhancing effect of inoculation density on shoot and
root growth has been reported in asparagus (Matsubara,
1973 and Matsubara and Clore, 1974) and potato (Sarkar
et al., 1997). This effect may be due to some growth
promoting substances diffusing from the explants. There
are several other factors which also influence the
proliferation rate in culture. These include the explant to
medium ratio, the explant to air ratio, the explant to
container volume ratio and type of container closures
(McClelland and Smith, 1990). The explant to medium
ratio would be responsible for influencing the microplant
proliferation rate in banana micropropagation.
Conclusion :
The cost of the in vitro shoot production of ‘Grand
Naine’ banana could be reduced by 33 per cent by
culturing at a density of 4 multiple bud clump/culture
bottles. Also cost of rooting could be lowered by 50 per
cent at density of 12 microshoots/culture bottle. It is
evident from the present study that the incubation of 4
multiple bud clumps and 12 microshoots in each culture
bottles is optimum for scaling-up of tissue culture
production of banana cv. ‘GRANDE NAINE’.
The calculated cost included only the variable items
of cost (MS salts, sucrose, hormones, electricity cost of
autoclave, laminar operation and incubation room and
wages of labour)
The calculated cost included only the variable items
of cost (MS salts, sucrose, hormones, electricity cost of
autoclave, laminar operation and incubation room and
wages of labour) .

13 Yearth
of Excellence
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